Abstract Excessive alcohol drinking-induced chronic liver disease has been recognized as a serious health problem in the United States. Increasing evidence suggests that inflammation plays an essential role in the development of alcoholic liver disease (ALD). In particular, the potent inflammatory response produced by the interactions between gut-derived lipopolysaccharide (LPS) and its cell surface receptor, Toll-like receptor 4 (TLR4), greatly contribute to alcohol-induced liver injury. However, the exact TLR4-expressing cell type that mediates this effect is largely unknown. Recently, we and others reported that hepatocyte TLR4 regulates obesity-related chronic inflammation, insulin resistance, and nonalcoholic liver disease. Furthermore, our preliminary data showed that mice lacking hepatocyte TLR4 had attenuated alcohol-induced early liver injury and reduced inflammation in white adipose tissue. In contrast, mice with TLR4 reactivation in hepatocyte accumulated more triglyceride content in the liver after chronic alcohol drinking. These findings support the potential role of hepatocyte TLR4 in mediating alcohol-induced steatohepatitis and insulin resistance. We will take advantage of our two unique mouse models that can selectively ablate and reactivate TLR4 expression in hepatocytes, respectively, to pursue the following specific aims. In specific aim 1, we will determine the role of hepatocyte TLR4 in the development of alcoholic steatohepatitis. We will use the mice lacking TLR4 expression specifically in hepatocyte to test the hypothesis that hepatocyte TLR4 is required for the development of alcoholic steatohepatitis induced by acute-on-chronic ethanol drinking. In specific aim 2, we will feed mice an ethanol-containing liquid diet chronically up to 8 weeks. We will test the hypothesis that hepatocyte TLR4 is required for the development of alcohol-induced insulin resistance. In specific aim 3, we will use a TLR4 reactivatable mouse model that can restore endogenous TLR4 expression specifically in hepatocytes to determine the sufficiency of hepatocyte TLR4 in alcohol-related steatohepatitis and insulin resistance. These studies will greatly enhance our knowledge of the regulatory role of hepatocyte TLR4 in alcoholic liver damage and associated metabolic disorders and facilitate the development of new anti-ALD therapies.